Oligonucleotides are essential reagents in many important molecular biology experiments, assays and information gathering operations, such as the polymerase chain reaction (PCR), diagnostic probes, single nucleotide polymorphism (SNP) detection, and genomic sequencing. The benefits of conducting the synthesis of oligonucleotides by the sequential addition and covalent attachment of monomeric units onto a solid support is well appreciated. In particular, the method of Caruthers is highly optimized and almost universally adopted (U.S. Pat. Nos. 4,458,066 and 4,973,679). The vast majority of the millions of oligonucleotides consumed each year are prepared by automated synthesis with phosphoramidite nucleoside monomers (Beaucage (1992) Tetrahedron Lett. 22:1859-62; U.S. Pat. No. 4,415,732).
The key step in oligonucleotide synthesis is the specific and sequential formation of internucleotide phosphate linkages between a 5′-OH group of one nucleotide and a 3′-OH group of another nucleotide. Accordingly, in the typical synthesis of oligonucleotides, the phosphate group of an “incoming” nucleotide is combined with the 5′-OH group of another nucleotide (i.e. the 5′-OH group is “phosphorylated” or “phosphitylated”). These groups must be capable of actively participating in the synthesis of the oligonucleotide. Thus, the 5′-OH groups are modified (typically with a dimethoxy trityl (“DMT”) group) such that an investigator can introduce two such nucleotides into a reaction chamber and adjust the conditions therein so that the two nucleotides are properly combined; by a series of successive such additions, a growing oligonucleotide having a defined sequence can be accurately generated.
The four bases of the nucleosides, adenine, thymine (uracil in the case of RNA), guanosine and cytosine, include moieties which are chemically reactive (e.g., exocyclic amino groups). These groups, unlike the 3′-OH and 5′-OH groups, must be “temporarily” protected, i.e. the protecting groups must be capable of blocking any reactive sites on the base until after the oligonucleotide synthesis is completed; after such synthesis is completed, these groups must also be capable of being removed from the bases such that the biological activity of the oligonucleotide is not affected.
The principal reason for temporarily protecting the base is that in the absence of such protecting groups, the exocyclic amino groups (“NH2”) of the bases can compete for binding to the 5′-OH group. If such a reaction takes place, the resulting product will not be useful. Accordingly, these protecting groups are important in reducing the occurrence of “side product formation” i.e. the formation of chemically similar, but unwanted, materials. To date, the most widely used protecting groups used in conjunction with the phosphoramidite methodologies for oligonucleotide synthesis are benzoyl for A and C, and isobutyryl for G and C, (thymine, which does not have an amino group, does not ordinarily require a protecting group).
Despite the structural similarities that iso-G shares with the standard bases, it has not been trivial to convert procedures and protecting groups used for the automated solid-phase synthesis of standard oligonucleotides to be suitable for preparing oligonucleotides containing iso-G. Accordingly, the synthesis of oligonucleotides and their analogs that employ the widely used protecting groups remains a tedious and costly process. There remains an ongoing need in this area for developing improved synthetic processes that facilitate the synthesis of oligonucleotides.